Flow rate balanced, dynamically adjustable sheath delivery system for flow cytometry

ABSTRACT

Disclosed is a sheath delivery system that uses a continuous flow of sheath fluid into a pressurized internal reservoir that substantially matches the outflow of sheath fluid through the nozzle of a flow cytometer. A substantially constant level of the sheath fluid is maintained. If the sheath fluid level falls below a desired level, or goes above a desired level, a dampened control system is used to reach the desired level. In addition, air pressure in the pressurized internal container is controlled so that an external sheath container can be removed and refilled with additional sheath fluid without stopping the sheath delivery system  100 . Differences in pressure are detected by a droplet camera, which measures the droplet breakoff point to determine the pressure of the sheath fluid in the nozzle.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 15/376,504, filed on Dec. 12, 2016, entitled “Flow RateBalanced, Dynamically Adjustable Sheath Delivery System for FlowCytometry,” which itself is a divisional application of U.S. patentapplication Ser. No. 13/918,156, filed on Jun. 14, 2013, entitled “FlowRate Balanced, Dynamically Adjustable Sheath Delivery System for FlowCytometry,” which claims the benefit under 35 U.S.C. § 119(e) to U.S.Provisional Patent Application No. 61/659,528, filed Jun. 14, 2012,entitled “Flow Rate Balance, Dynamically Adjustable Sheath DeliverySystem for Flow Cytometry,” which are all incorporated herein byreference for all that they disclose and teach.

BACKGROUND

Flow cytometers are useful devices for analyzing and sorting varioustypes of particles in fluid streams. These cells and particles may bebiological or physical samples that are collected for analysis and/orseparation. The sample is mixed with a sheath fluid for transporting theparticles through the flow cytometer. The particles may comprisebiological cells, calibration beads, physical sample particles, or otherparticles of interest. Sorting and analysis of these particles canprovide valuable information to both researchers and clinicians. Inaddition, sorted particles can be used for various purposes to achieve awide variety of desired results.

SUMMARY

An embodiment of the present invention may therefore comprise a methodof controlling pressure of a sheath fluid in a pressurized container ina flow cytometer comprising: continuously pumping the sheath fluid froman external container into the pressurized container to attempt tomaintain a substantially constant sheath fluid level in the pressurizedreservoir so that an in-flow rate of sheath fluid flowing into thepressurized reservoir is substantially equal to an out-flow rate of thesheath fluid flowing out of the pressurized reservoir; adjusting thein-flow rate of the sheath fluid flowing into the pressurized reservoirwhenever the substantially constant sheath fluid level changes.

An embodiment of the present invention may further comprise a method ofreplacing an external sheath container while continuously operating asheath fluid system in a flow cytometer comprising: substantiallymatching an output flow rate of an internal reservoir of sheath fluidflowing through a nozzle with an input flow rate of an input flow ofsheath fluid from the external sheath container to the internal sheathcontainer to substantially maintain a preselected level of sheath fluidin the internal reservoir; stopping the input flow of the sheath fluidwhile the external sheath container is removed; increasing air pressurein the internal reservoir while the input flow of the sheath fluid isstopped to substantially maintain a constant pressure on the sheathfluid through the nozzle; replacing the external sheath container;pumping sheath fluid from the external sheath container to the internalreservoir at an input flow rate that is greater than the output flowrate until the sheath fluid in the internal reservoir reaches thepreselected level; reducing the air pressure in the internal reservoirwhile the internal reservoir is being filled to the preselected level tomaintain a substantially constant pressure on the sheath fluid flowingthrough the nozzle.

An embodiment of the present invention may further comprise a sheathfluid system for supplying sheath fluid in a flow cytometer at asubstantially constant pressure comprising: an internal pressurizedsheath fluid reservoir that supplies the sheath fluid to a nozzle; anexternal sheath fluid container that supplies the sheath fluid to theinternal pressurized sheath fluid reservoir, and that can be removed forresupplying the sheath fluid to the sheath container; a pump thatcontinuously supplies the sheath fluid from the external sheath fluidcontainer to the internal pressurized sheath fluid container to maintaina level of the sheath fluid in the internal pressurized reservoirsubstantially constant by substantially matching an in-flow rate of thesheath fluid from the external sheath fluid container to the internalpressurized sheath fluid reservoir with an out-flow rate of the sheathfluid from the internal pressurized sheath fluid reservoir, unless theexternal sheath fluid container has been removed for resupplying thesheath fluid; a compressor that supplies a source of compressed air; anair regulator that regulates the compressed air that is connected to theinternal pressurized sheath fluid reservoir to supply regulatedpressurized air to the internal pressurized sheath fluid reservoir; anair pressure controller that controls the regulated pressurized air, andincreases the pressure of the regulated pressurized air whenever thelevel of the sheath fluid in the internal pressurized sheath fluidreservoir falls below a preselected level, and decreases the pressurewhenever the level is increasing, so as to maintain a substantiallyconstant pressure and a substantially constant velocity of the sheathfluid that exits the internal pressurized sheath fluid reservoir.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of one embodiment of a sheathdelivery system.

FIG. 2 is a flow diagram illustrating a process for maintaining asubstantially constant sheath height.

FIG. 3 illustrates images of the breakoff point of droplets from streamsthat indicate the velocity of the streams.

DETAILED DESCRIPTION OF THE EMBODIMENTS

FIG. 1 is a schematic illustration of a sheath delivery system 100. Thesheath delivery system 100 includes an external sheath container 102that provides sheath fluid to an internal pressurized reservoir 104. Asdisclosed below, the external sheath container 102 can be removed by auser and refilled or replaced without stopping the operation of thesheath delivery system 100, while maintaining a substantially constantpressure on the sheath fluid that is delivered through nozzle 112.

For flow cytometers to operate properly, it is important that the stream126, through nozzle 112 has a consistent velocity, which is dependentupon the pressure of the sheath fluid 144 in the sheath delivery tube124. Otherwise, the flow cytometer must be continuously calibrated. Somesystems that supply sheath fluid in flow cytometers have utilized largetanks to avoid the problem of shutting down the system when additionalsheath fluid is needed. These large tanks are heavy and expensive.Furthermore, the change in the fluid height in these large tanks duringoperation results in considerable pressure changes between a full andnearly empty container. The pressure of the fluid that is supplied tothe nozzle is the pressure supplied by pressurized air in the tank andthe pressure that is supplied by the height of the fluid in the tank. Insome systems, the level of the fluid can change by as much as twelveinches between a full and nearly empty container. This is a change ofapproximately 0.5 psi. If the sheath pressure is approximately 30 psi,the change in pressure resulting from the fluid can be as much as a 1.7percent change in the pressure of the sheath fluid delivered to thenozzle. Additionally, air regulators that supply pressurized air to thetank may drift over time, and the air pressure in a pressurized tank maychange, which also changes the pressure of the sheath fluid that isdelivered to the nozzle. Some systems have utilized external sheathcontainers and pumps to pump the sheath fluid into an internalpressurized chamber for use during a short period of operation. However,such systems must be stopped during this filling procedure, whichresults in short run times and may necessitate recalibration of thesystem. These systems also do not account for changes in the pressure ofthe sheath fluid in the nozzle due to changes in the depth of the sheathfluid in the internal container. Other systems have attempted to resolvethe short run time issue by using float switches in the internalcontainer that turn a pump on and off to allow sheath fluid from anexternal sheath reservoir to flow into the internal container when thelevel drops by a predetermined amount in the internal container.However, this still results in intermittent, abrupt changes in thepressure of the sheath fluid flowing through the nozzle due to the leveldifferences in the internal container, as a result of the non-continuousoperation of the pump.

The embodiment of FIG. 1 operates using a continuous flow of sheathfluid into the pressurized internal reservoir 104 from the externalsheath container 102. The sheath fluid provided by the external sheathcontainer 102 is pumped at a rate that substantially matches theout-flow of the sheath fluid 144 through nozzle 112. In addition, if thelevel 145 of sheath fluid falls in the pressurized internal containerbecause the flow rates are not matched, the in-flow of sheath fluid isslowly changed to make up for changes in the level 145 of the sheathfluid 144 in the pressurized internal container 104. By using acontinuous in-flow of sheath fluid 142 from the external sheathcontainer 102 that substantially matches the out-flow of sheath fluid142 through nozzle 112, intermittent variations in the pressure of thesheath fluid 144 in the nozzle 112 do not occur.

The control loop (FIG. 2) for the process of matching flow rates betweenthe input to the pressurized internal reservoir 104, and the out-flow tonozzle 112, are tightly controlled and dampened, so that pressurechanges from the level 145 of the sheath fluid 144 are negligible. Inaddition, the control of the pressurized air 146 further reducesvariations in the pressure of the sheath fluid 144 flowing throughnozzle 112, as described in more detail below.

The system illustrated in the embodiment of FIG. 1 can utilize twocontrol systems that separately control the level 145 of the sheathfluid 144 in the internal container 104, and the air pressure of thepressurized air 146 that is supplied by compressor 118 and air regulator108. Utilizing these two control systems, a substantially consistentpressure of the sheath fluid can be provided to the nozzle 112. Sincethe two control loops utilize feedback from different sources, i.e., thelevel 145 of sheath fluid 144 and the velocity of the sheath fluidstream 126, the two separate systems can work in concert toautomatically provide a substantially constant pressure of sheath fluidto the nozzle 112. The control system that controls the air pressure inthe pressurized internal reservoir may be used either on a constantbasis in concert with the level control system, or simply when there isa hot swap of the external sheath container, as explained in more detailbelow.

As illustrated in FIG. 1, sheath pump 106 pumps sheath fluid 142 intothe pressurized internal reservoir 104 to provide a supply of sheathfluid 144 in the pressurized internal reservoir 104. Air regulator 108provides regulated air 120 to the pressurized internal reservoir 104 toproduce a supply of pressurized air 146 in the pressurized internalreservoir 104. Pressurized air tube 140 delivers the pressurized airfrom the air regulator 108 to the pressurized internal reservoir 104.Compressor 118 provides the compressed air 119 to the air regulator 108.Sheath supply tube 134 supplies the sheath fluid 142 to the sheath pump106. Pressurized sheath tube 136 provides the sheath fluid from thesheath pump 106 to the pressurized internal reservoir 104. Levelcontroller/monitor 110 comprises an electronic controller that generatesa pump speed control 132 that is applied to the sheath pump 106. Levelcontroller/monitor 110 receives a level sensor signal 111 from the levelsensor 138. Level sensor 138 can comprise any type of level sensor. Asillustrated in the embodiment of FIG. 1, level sensor 138 comprises anultrasonic detector that is disposed at the bottom of the pressurizedinternal reservoir 104 which measures the height of the sheath fluid 144in the pressurized internal container 104 with a 0.5 mm resolution. Thelevel sensor signal 111 generated by level sensor 138 is applied to thelevel controller/monitor 110, which controls the sheath pump 106 viapump control speed signal 132 to maintain a substantially constant levelof the sheath fluid 144 in the pressurized internal reservoir 104 in themanner described with respect to FIG. 2.

Sheath uptake tube 122, illustrated in FIG. 1, provides the pressurizedsheath fluid 144 from the bottom of the pressurized internal reservoir104 and delivers the pressurized fluid through the sheath delivery tube124 to the nozzle 112. In one example, the pressurized air 146 may bepressurized to approximately two atmospheres, which is approximately 30psi. The pressure of the pressurized air 146 is added to the pressure ofthe sheath fluid 144, which is dependent upon the level 145 of thesheath fluid 144 in the pressurized internal reservoir 104. In roundnumbers, one atmosphere is about 15 psi. The sheath fluid 144, in roundnumbers, provides a pressure of about ½ psi per each foot of depth ofthe sheath fluid 144. Accordingly, the pressure of the sheath fluid 144in the sheath delivery tube 124 is the pressure of the pressurized air146 together with the pressure created by the sheath fluid 144 inaccordance with the level 145 of the sheath fluid 144 in the pressurizedinternal reservoir 104 minus any changes in fluid height.

A substantially constant pressure of the sheath fluid 144 in the sheathdelivery tube 124 of FIG. 1 can be achieved by carefully maintaining asubstantially constant level 145 of the sheath fluid 144 in thepressurized internal reservoir 104, as well as maintaining asubstantially constant pressure of the pressurized air 146. Air pressurecontroller 116 generates an air pressure control voltage 130 thatoperates the air regulator 108. The regulated air 120 is supplied to thepressurized internal reservoir 108 via the pressurized air tube 140.Droplet camera 114 determines the position of the bottom of the stream,which corresponds to the location of the breakoff point of the dropletsfrom stream 126 into drops 128. As explained in more detail with respectto FIG. 3, the location of the breakoff point of the drops 128 isindicative of the velocity of the stream 126, which is dependent uponthe pressure of the sheath fluid 144 that is delivered to nozzle 112.The droplet camera 114 provides the graphic data to the air pressurecontroller 116 that processes that image data to generate the airpressure control voltage 130. Air pressure controller 116 generates anair pressure control voltage 130 to compensate for drift in the airpressure of the pressurized air 146 due to drift of the operation of theair regulator 108 and compressor 118, as explained in more detail withrespect to FIG. 3.

As also illustrated in FIG. 1, a three-way valve 148 is connected to thepressurized sheath tube 136. During normal operation, the three-wayvalve 148 causes sheath fluid 142, from external sheath container 102,to be directed into the pressurized internal reservoir 104 viapressurized sheath tube 136. However, bubbles may form in the sheathsupply tube, causing an airlock in the sheath pump 106. Airlocks of thesheath pump 106 may be created when air bubbles enter the sheath supplytube 134. This may occur when the external sheath container 102 isremoved from the system and refilled with sheath fluid 142 or, theexternal sheath container 102 is replaced with a fill container, duringa process referred to as a “hot swap,” which is described in more detailwith respect to FIG. 3. Since the sheath pump 106 is pumping the sheathfluid 142 into pressurized internal reservoir 104, that has a pressureon the order of 2 atmospheres, any air bubbles that enter the sheathpump 106 from the sheath supply tube 134 can easily create an airlock inthe sheath pump 106. In order to clear the airlock, the three-way valve148 is switched, so that the fluid from the sheath pump is directed intoa waste disposal 150 that is at the ambient atmospheric pressure. Sheathpump 106 has sufficient power to clear an airlock by pumping the sheathfluid 142 into an ambient pressure waste disposal 150, but may not havesufficient power to clear airlocks into the multiple atmospherepressurized air 146. Hence, by directing the output of the sheath pump106 to an ambient atmospheric pressure, the airlock can be cleared.

Also, as illustrated in FIG. 1, if the level controller/monitor receivesa level sensor signal 111 from the level sensor 138 that indicates thatthe level 145 of the sheath fluid 144 in the pressurized internalreservoir 104 is going down at a rate that is more than should beobserved by the level sensor 138 for the rate at which the sheath pump106 is being operated, the three-way valve 148 is activated to clear anairlock. In other words, the sheath pump 106 is provided a pump speedcontrol 132, which is a voltage that is a percentage of the full voltageat which the sheath pump 106 operates. A comparison of the voltage ofthe pump speed control 132 with the level sensor signal 111 can indicatethat an airlock may exist in the sheath pump 106, which can be used totrigger the level controller/monitor 110 to generate the purge controlsignal 152.

FIG. 2 is a flow chart 200 that illustrates the process for maintaininga substantially constant level 145 of the sheath fluid 144. At step 202,the process starts. At step 204, the internal reservoir is pumped ordrained to a preselected level, such as 300 ml. This occurs prior to thestart of operation of the flow of sheath fluid 144 through nozzle 112.At step 206, the sheath pump 106 is started and the compressor 118 isstarted, to raise the level of the pressurized air 146 to a desiredpreset level. At this point, fluid sheath 144 begins to flow through thenozzle. Almost simultaneously, at step 208, the sheath pump 106 is setto a default pump speed, based upon an empirically derived flow rate ofthe nozzle at the selected level 145 of sheath fluid 144 and airpressure of pressurized air 146. In one example, the flow rate throughthe nozzle 112 is estimated to be 8 mL/min at a level of 300 mL and apressure of 30 psi. The default pump speed is selected in an attempt tomatch the flow rate through the nozzle, i.e., 8 ml/min. In that regard,empirical data regarding the flow rate of nozzle 112 can be collected.Of course, other ways of initially estimating the amount of sheath fluiddelivered by the nozzle 112 can be used to set the default pump flowrate to substantially match the rate of flow of sheath fluid throughnozzle 112.

The process of setting the default pump speed, at step 208 of FIG. 2,involves the use of Equation 1.

Pump control percentage=(pump rate*constant related to pumpoperation)+offset voltage of pump.  (Equation 1)

The pump control percentage is a percentage of the full operation of thepump. With the operating range of a pump, most pumps have a linearresponse to applied voltage levels. However, most pumps have an offsetvoltage. The offset voltage is the voltage at which the pump starts tooperate and pump fluid. For example, the sheath pump 106 utilized in theembodiment of FIG. 1, does not start pumping until ten percent of thefull operating voltage of the pump is applied to the pump. For example,in an example of the embodiment of FIG. 1, the full operating voltage ofthe sheath pump 106 is 5 volts. From empirical data, it was determinedthat sheath pump 106 starts pumping fluid when 0.5 volts is applied tothe sheath pump 106. The voltage of 0.5 volts is ten percent of the fivevolts that causes the sheath pump 106 to operate at full capacity.Hence, the offset of sheath pump 106 is 10%. From 0.5 volts to 5.0volts, the output of the sheath pump 106 is substantially linearlyrelated to the voltage. Because of this linear relationship, equation 1can be correlated to the standard Equation 2.

y=mx+b  (Equation 2)

In equation 2, b is the offset, which was empirically determined to be10%. The slope of the curve (m) for the sheath pump 106 of FIG. 1 canalso be empirically determined and was found, in one example, to be 1.25from data collected by operating the sheath pump 106. This results inEquation 3, which is created from the collected empirical data forsheath pump 106 of the embodiment of FIG. 1.

Pump Control Percentage=1.25*Pump Flow Rate+10  (Equation 3)

From Equation 3, it can be determined that an initial default pump flowrate of 8 mL/min results in a pump control percentage of 20%, whichequates to 1 volt that should be applied to sheath pump 106. Other pumpshave different characteristics and empirical data must be collected foreach pump to verify Equation 3. However, it can be assumed that pumpsfrom the same manufacturer, with the same model number, may have verysimilar operating characteristics, such that Equation 3 is most probablyvalid for same make and model number pumps. Initial default pump floatrates can normally be used for pumps that are the same make and model,so that empirical data does not have to be collected for each pump.

At step 210 of FIG. 2, the process is delayed for a preset period, forexample, 5.0 seconds. This delay is used to allow the flow rate tostabilize. The level 145 of the sheath fluid 144 is measured in thepressurized internal reservoir 104 at step 212. To suppress noise in thereadings of level 145 of the sheath fluid 144, in one embodiment, 42readings of the level sensor 138 are taken, the lowest five and thehighest five readings are discarded and the mean value of the remaining32 readings is used as the level 145 of the sheath fluid. This processremoves noise and aberrant readings. After the level has been measured,the system delays for a second predetermined time period at step 214.The system illustrated in the embodiment of FIG. 1 delays for a secondtime period of 30 seconds. This delay is somewhat substantial so that atrend can be observed in the level 145 of the sheath fluid 144. At theend of the second predetermined time period, a new level 145 of thesheath fluid is measured at step 216. At step 218, a new pump speed iscalculated. In order to calculate a new pump speed, the differentialactual outflow of fluid must be calculated. Assuming the level hasdecreased, Equation 4 calculates the differential actual outflow asfollows:

Differential Actual Outflow=Pump Rate+Level Decrease*2  (Equation 4)

The level decrease is multiplied by two since the change in fluid leveloccurs over a 30 second period, and the data is indicated on a perminute basis.

For example, if the initial pump speed is set at 8 mL per minute and thelevel decreases by 0.25 mL in the 30 second delay period, the truedifferential outflow during that period is 8.5 mL per minute, which iscalculated as:

Differential Actual Outflow=8.0(the pump rate)+0.25(the leveldecrease)*2=8.5   (Equation 5)

The new pump speed is the modified pump speed that is calculated toslowly reduce the difference in the level 145 of the sheath fluid 144from the desired level. In order to calculate the new pump speed,Equation 6 should be used:

New Pump Speed=Differential Actual Outflow+Error/2  (Equation 6)

The error is calculated as the difference between the desired levelminus the new level. If the desired level is 300 mL and the new level is299 mL, the error is equal to 1 mL. The system attempts to return to thenew level within a period of 2 minutes, even though the sampling rate isevery 30 seconds. That accounts for the “2” in Equation 6. At the end ofevery 30 second period, a new pump speed is calculated based upon a pumpspeed that would return the new level to the desired level in a 2 minuteperiod. In this manner, the target of the new pump speed will not beovershot and the control system is adequately dampened to provide a newpump speed that will slowly return to the desired level. In the examplegiven above, the ideal level was 1 mL low, which is the error. Theerror, 1 mL divided by 2 minutes equals 0.5 mL/minute. Inserting thesevalues in Equation 6:

New Pump Speed=8.5+0.5=9.0 mL/minute  (Equation 7)

The 9.0 mL/minute is then converted to a pump control percentage usingthe pump control equation (Equation 3). In this case:

Pump Control Percentage=1.25×9.0+10=21.25%  (Equation 8)

The actual voltage that is applied to the pump is given as follows:

Voltage Applied to Pump=21.25%×5.0 volts=1.0625 volts  (Equation 9)

The process of FIG. 2 then returns to step 210 and delays for the firstpreset period.

Of course, other types of controllers can be used, such as standard PIDcontrollers. Proportional integral derivative controllers (PIDcontrollers) use a generic control feedback that is widely used inindustrial control systems. A PID controller calculates an error valueas the difference between a measured process variable and a desired setpoint. The controller attempts to minimize the error by adjusting theprocess control inputs. The PID controller calculation involves threeseparate parameters that comprise the proportional, the integral and thederivative values. Heuristically, these values can be represented interms of time wherein P depends on the present error, I depends on theaccumulation of past errors and D is a prediction of future errors,based on the current rate of change. A weighted sum of these threevalues is used to adjust the process, in this case, the voltage appliedto sheath pump 108. The accuracy of the PID controllers is very muchdependent upon the weighting of each of the PID values.

FIG. 3 illustrates images 302, 304, 306 of the breakoff point ofdroplets 310, 314, 318, from streams 309, 312, 316, respectively, thatare taken by the camera 114 and sent to the air pressure controller 116.As illustrated in FIG. 3, an image 302 shows droplets 310 breaking awayfrom stream 309 at a reference line 308. Reference line 308 isconsidered a reference for a desired stream velocity that is produced bystream 309. A slower velocity stream 312 is illustrated by image 304,which is the result of a pressure decrease of the stream 312, such thatthe breakoff point of the droplets 314 from the stream 312 is above thereference line 308 at a higher location. Image 306 shows stream 316,which has a higher velocity, which is the result of a higher pressure onstream 316, resulting in the droplets 318 breaking off from stream 316at a lower point below the reference line 308. As such, the velocity ofthe stream and the resultant pressures on the streams can be determinedby identifying the breakoff point of the droplets from the stream. Asdescribed above, air regulator 108 sets the pressure of the pressurizedair 146 in the pressurized internal reservoir 104. Since air regulatorsmay tend to drift over time and with temperature during operation, thedroplet camera 114 can be used to accurately determine if there is achange in air pressure based upon the velocity of the stream, asindicated by the images 302, 304, 306 provided by the droplet camera114. A strobe light may be used with the droplet camera 114 that has afixed phase relative to the sign wave of a piezoelectric vibrator (notshown) that is used to create droplets, as illustrated in FIG. 3. Theair pressure controller 116 operates by storing the location of thereference line 308. When the bottom of the stream moves below thereference line 308, as indicated by higher velocity stream 312, there isa pressure increase and the pressure of the pressurized air 146 isdecreased by 0.01 psi per second. Likewise, if the bottom of the streammoves above the reference line 308, the pressure of the pressurized air146 is increased by 0.01 psi every second. These changes are slow movingchanges in the pressurized air 146 that do not radically change thepressure, which could negatively affect the sorting process. Of course,other types of controllers can be used including different values. Forexample, a PID controller could be used to calculate appropriate changesin air pressure.

Since the embodiment of FIG. 1 is able to control the pressure of thepressurized air 146, the external sheath container 102 can be removedand refilled without stopping the sheath delivery system 100 of FIG. 1.Removal of the external sheath container 102 for refilling is referredto as a hot swap mode, since the sheath fluid system continues tooperate. In the hot swap mode, the sheath pump 106 is stopped completelyfor the period of time that it takes to remove the external sheathcontainer 102, refill the external sheath container 102, and replace theexternal sheath container 102 in the sheath delivery system 100 ofFIG. 1. The user of the sheath delivery system 100 removes the externalsheath container 102 and either fills the external container or replacesthe external container with a new external container that is filled withsheath fluid. With an average outflow of 8 mL per minute, the sheathfluid level will slowly decrease in the internal pressurized containerduring the hot swap mode. The change in pressure from the sheath fluid144 is compensated for by increasing the pressure of the pressurized air146. The lower pressure of the sheath fluid during the hot swap mode isdetected as the slower velocity image 304. When the slower velocityimage 304 is detected, the air pressure controller 116 continues toincrease the air pressure at a rate of 0.01 psi per second by applyingthe air pressure control voltage 130 to the air regulator 108. The airpressure in the internal pressurized sheath fluid reservoir continues toincrease until a new bottle of sheath fluid or refilled bottle of sheathfluid 142 is placed in the sheath delivery system 100. Once the newbottle is reattached, the user can exit the hot swap mode. At thatpoint, the sheath pump 106 is restarted, and sheath fluid 142 is pumpedinto the pressurized internal reservoir 104 at a rate that is calculatedby the level of the controller/monitor 110 in the manner set forthabove. As the sheath fluid enters the pressurized internal reservoir104, the air pressure controller 116 slowly reduces the pressure of thepressurized air 146 in the internal reservoir 104 to maintain asubstantially constant pressure on the sheath fluid 144 exiting internalreservoir 104 that travels through the nozzle 112. In this manner, thesheath pressure of the sheath fluid 144 that flows through the nozzle112 is carefully regulated within the requirements for stable sorting toprovide a substantially constant pressure.

The foregoing description of the invention has been presented forpurposes of illustration and description. It is not intended to beexhaustive or to limit the invention to the precise form disclosed, andother modifications and variations may be possible in light of the aboveteachings. The embodiment was chosen and described in order to bestexplain the principles of the invention and its practical application tothereby enable others skilled in the art to best utilize the inventionin various embodiments and various modifications as are suited to theparticular use contemplated. It is intended that the appended claims beconstrued to include other alternative embodiments of the inventionexcept insofar as limited by the prior art.

What is claimed is:
 1. A method of adding sheath fluid to a flowcytometer having a positively pressurized reservoir holding both avolume of sheath fluid and a volume of positively pressurized air,wherein the flow cytometer is configured to flow sheath fluid from thepositively pressurized reservoir through a nozzle at an out-flow rate,the method comprising: pumping, using a pump interposed between anexternal container containing sheath fluid and the positivelypressurized reservoir, the sheath fluid from the external container intothe positively pressurized reservoir at an in-flow rate; determining theout-flow rate of fluid flowing out of the nozzle based on data from avisual sensor configured to detect droplet locations of the sheath fluidflowing out of the nozzle; reducing the in-flow rate of the sheath fluidflowing into the positively pressurized reservoir to zero; andincreasing, after the reducing and based upon the determination, thepressure of the volume of positively pressurized air so that theout-flow rate of fluid flowing out of the nozzle, as determined by thevisual sensor, remains substantially constant.
 2. The method of claim 1,further comprising replacing, after the reducing and during theincreasing, the external container with a new external container.
 3. Themethod of claim 2, wherein the replacing further comprises: fluidicallydisconnecting the external container from the pump, and fluidicallyconnecting the new external container to the pump.
 4. The method ofclaim 1, further comprising refilling, after the reducing and during theincreasing, the external container with the sheath fluid.
 5. The methodof claim 4, wherein the refilling further comprises flowing sheath fluidinto the external container.
 6. The method of claim 5, wherein therefilling further comprises: fluidically disconnecting the externalcontainer from the pump, and fluidically connecting the externalcontainer to the pump.
 7. The method of claim 1, further comprising:increasing, after the reducing, the in-flow rate of the sheath fluidflowing into the positively pressurized reservoir to a non-zero value bypumping, using the pump, the sheath fluid from the external containerinto the positively pressurized reservoir, and adjusting, during theincreasing of the in-flow rate of the sheath fluid and based upon thedetermination, the pressure of the volume of positively pressurized airso that the out-flow rate of fluid flowing out of the nozzle, asdetermined by the visual sensor, remains substantially constant.
 8. Themethod of claim 7, wherein the adjusting further comprises reducing thepressure of the volume of positively pressurized air in the positivelypressurized reservoir.
 9. The method of claim 7, wherein the increasingof the in-flow rate of the sheath fluid further comprises increasing thein-flow rate of the sheath fluid flowing into the positively pressurizedreservoir to an in-flow rate that is greater than the out-flow rate ofthe fluid flowing out of the nozzle.
 10. The method of claim 7, furthercomprising determining a level of the sheath fluid in the positivelypressurized reservoir, wherein the increasing of the in-flow rate of thesheath fluid is based on the determination of the level of the sheathfluid in the positively pressurized reservoir.
 11. The method of claim10, further comprising adjusting, after the increasing of the in-flowrate of the sheath fluid, the in-flow rate of the sheath fluid flowinginto the positively pressurized reservoir so that the out-flow rate offluid flowing out of the nozzle, as determined by the visual sensor,remains substantially constant.
 12. The method of claim 11, wherein theadjusting of the sheath fluid flowing into the positively pressurizedreservoir further comprises decreasing the in-flow rate of the sheathfluid flowing into the positively pressurized reservoir.
 13. The methodof claim 11, wherein the adjusting is based on the determination of thelevel of the sheath fluid in the positively pressurized reservoir. 14.The method of claim 10, wherein the determining the level of the sheathfluid in the positively pressurized reservoir further comprises: takinga plurality of measurements of a height of the sheath fluid in thereservoir, and determining whether a trend in the height of the sheathfluid has been observed.
 15. The method of claim 7, wherein theincreasing of the in-flow rate of the sheath fluid further comprisesrestarting the pump.
 16. The method of claim 1, wherein the reducingfurther comprises stopping the pump.
 17. The method of claim 1, furthercomprising: detecting a vertical location of a breakoff point at whichdroplets separate from a stream of fluid exiting the nozzle of the flowcytometer; comparing the vertical location with a desired verticallocation; and adjusting the pressure of the volume of positivelypressurized air in the positively pressurized reservoir to cause thevertical location of the breakoff point to substantially match thedesired vertical location.
 18. The method of claim 17, wherein detectingthe vertical location of the breakoff point comprises using a dropletcamera to record images of the stream.